1. Field of the Invention
The present invention relates to the field of molecular biology and nucleic acid chemistry. More specifically, it relates to methods and reagents for quantitating expression of mRNA that encodes an active form of hTERT, the catalytic subunit of telomerase, as an indicator of the presence of cancerous cells.
2. Description of Related Art
Telomerase is an RNA-dependent DNA polymerase that synthesizes telomeric DNA. The core enzyme consists of an RNA component that serves as a template for the synthesis of telomeric repeats, and a catalytic subunit with reverse transcriptase activity, designated hTERT (also referred to as hTCS1, hTRT, and hEST2).
Assays of telomerase activity are described in, for example, International Patent Publication No. WO 97/15687, incorporated herein by reference. A PCR-based telomeric repeat amplification protocol (TRAP) is described in Chang, 1999, in PCR Applications (Innis et al., eds.) Chapter 17, Academic Press, San Diego; Kim et al. 1994, Science 266:2011-2015; and U.S. Pat. No. 5,629,154, each incorporated herein by reference. Telomerase activity is not detected in most human somatic cells, but is detected in immortalized cell lines and in human tumors. The detection of elevated telomerase activity in a tissue sample can be used to identify cancerous tissues.
Nakamura et al., 1997, Science 277:955-959, identified the gene encoding the catalytic subunit of human telomerase (designated therein hTRT). They reported that hTRT mRNA was not expressed in telomerase-negative cell lines, but was expressed in telomerase-positive immortal cell lines, and concluded that the expression of mRNA from the human gene correlates with telomerase activity.
Meyerson et al., 1997, Cell 90:785-795, described the cloning of the gene encoding the catalytic subunit of telomerase (designated therein hEST2) and report that it is expressed at high levels in primary tumors, cancer cell lines, and telomerase-positive tissues, but is undetectable in telomerase-negative cell lines and differentiated telomerase-negative tissues. They report that, although they found a general correlation between hEST2 mRNA levels and telomerase activity, these two measures where not present in a constant, predictable ratio. Consequently, Meyerson et al. speculated that other mechanisms besides the modulation of mRNA levels may be important in the regulation of telomerase activity.
Kilian et al., 1997, Human Molecular Genetics 6(12):2011-2019, identified the gene encoding the catalytic subunit of human telomerase (designated therein hTCS 1). They reported that, although the gene is present in a single copy, it is expressed in a complex splicing pattern that gives rise to a number of potential proteins. A number of different transcript sequence variants were identified. By comparing the splice variants with a reference sequence essentially identical to that reported by Nakamura et al., supra, they identified two splice variant deletions, a 36 nucleotide deletion designated xcex1 and a 182 nucleotide deletion designated xcex2, as well as 3 different insertions. Deletion of the xcex1 region was reported as resulting in a small in-frame 12 amino acid deletion. The xcex2 region deletion was reported to encode a truncated protein.
The present invention provides reagents, methods, and kits for the quantitation of expression of hTERT mRNA that encodes an active hTERT protein. The reagents, methods, and kits provide and improvement over methods previously described by enabling a more accurate estimate of telomerase activity.
The level of hTERT mRNA expression provides information that assists in the diagnosis of cancers. Telomerase activity, repressed in most normal somatic cells, is reactivated in immortal tumor cells. We have confirmed that telomerase activity is regulated at the level of gene expression, and that the level of hTERT mRNA expression provides a measure of telomerase activity. The present invention provides an accurate and reproducible measure of telomerase activity by selectively measuring mRNA that encodes an active hTERT protein.
The hTERT gene consists of 16 exons and 15 introns spanning about 35 kilobases and encodes 1132 amino acids. Several splice variants have been reported. We have determined that mRNA splice variants which encode an active hTERT protein can be discriminated from the predominant splice variants which encode inactive forms of the hTERT protein based on the presence the xcex2 region, the 182 nucleotide deletion encompassing exon 7 and exon 8. The methods of the present invention selectively measure only hTERT mRNA containing the xcex2 region and, thus, enable quantitation of essentially only mRNA encoding an active hTERT protein.
One aspect of the invention is the discovery of a previously unobserved splice variant in which there is a deletion of a subregion of the xcex2 region which, most likely, corresponds to a deletion of exon 8, specifically. The occurrence of this splice variant represent a previously unrecognized problem in estimating telomerase activity based on hTERT mRNA expression. The present invention provides a solution to this previously unrecognized problem.
One aspect of the invention relates to methods and reagents for quantitating hTERT mRNA in a human sample. The methods and reagents selectively measure primarily mRNA encoding the active form of the hTERT protein, which provides a more accurate surrogate measure of telomerase activity and, thereby provides an improved marker for use in cancer diagnosis.
The methods of the invention involve amplifying a target hTERT mRNA sequence using a pair of primers in which one primer hybridizes to a sequence within exon 8, which is a subregion of the xcex2 region, and the other primer hybridizes to a sequence outside the xcex2 region, preferably within exon 6, upstream of the xcex2 region, and quantitatively detecting the formation of amplification products. Such a primer pair has the property that the amplified mRNA corresponds to primarily mRNA that encodes an active hTERT protein. Particularly preferred amplification primers are described below. The preferred primers are particularly advantageous in that they provide for the specific and efficient amplification of the mRNA sequence.
The detection of amplification can be carried out using a variety of methods, as described below. In a preferred embodiment, the amplified hTERT mRNA sequence is detected by probe hybridization. In a particularly preferred embodiment, the amplified product is detected using a probe which hybridizes to an mRNA sequence at least partially within exon 7, more preferably encompassing the exon7/exon 8 splice junction. Such a probe has the property that it would enable the detection of a splice variant in which only exon 7 is deleted, which has yet to be observed. An exon 7 deletion splice variant would be identified by successful amplification of a product not that did not hybridize to the probe.
Another aspect of the invention relates to methods and reagents for determining the telomerase activity in a human sample, which involves quantitating hTERT mRNA using the methods and reagents of the present invention. The quantity of hTERT mRNA, when calibrated as described herein, provides an estimate of the telomerase activity. The present invention, by measuring essentially only hTERT mRNA which encodes an active hTERT protein, provides a more accurate estimate of telomerase activity.
Another aspect of the invention relates to methods of identifying the presence of cancerous cells in a tissue sample which involves detecting an increased level of hTERT mRNA that encodes an active form of the hTERT protein or, equivalently, an increased level of telomerase activity, using the methods and reagents of the present invention.
Another aspect of the invention relates to oligonucleotides useful as amplification primers or detection probes in the methods of the present invention.
Another aspect of the invention relates to kits useful for quantitating hTERT mRNA comprising one or more of the reagents of the present invention. These kits take a variety of forms. In one embodiment, the kits of the inventions comprise primers and, optionally, probes, as described above. The kits can also comprise one or more amplification reagents, e.g., primers, polymerase, buffers, and nucleoside triphosphates.